Amines and methods of making and using the same

ABSTRACT

Novel amine compounds are provided by the present invention. Methods of preparing and using said novel amine compounds are also provided.

This application is a continuation of application Ser. No. 07/943,516,filed Sep. 11, 1992, now abandoned, which is a continuation-in-part ofapplication Ser. No. 07/558,663, files Jul. 27, 1990 (now U.S. Pat. No.5,138,045) and of application Ser. No. 07/844,845, filed Mar. 3, 1992(now U.S. Pat. No. 5,218,105).

FIELD OF THE INVENTION

This invention relates to novel amine-containing compounds useful fortherapeutics and methods of making and using the same.

BACKGROUND OF THE INVENTION

It is well known that most of the bodily states in mammals includingmost disease states, are effected by proteins. Such proteins, eitheracting directly or through their enzymatic functions, contribute inmajor proportion to many diseases in animals and man. Classicaltherapeutics has generally focused upon interactions with such proteinsin efforts to moderate their disease causing or disease potentiatingfunctions. Recently, however, attempts have been made to moderate theactual production of such proteins by interactions with molecules thatdirect their synthesis, intracellular RNA. These interactions involvedthe binding of complementary "antisense" oligonucleotides or theiranalogs to the transcellular RNA in a sequence specific fashion such asby Watson-Crick base pairing interactions.

The pharmacological activity of antisense compounds, as well as othertherapeutics, depends on a number of factors that influence theeffective concentration of these agents at specific intracellulartargets. One important factor is the ability of antisense compounds totraverse the plasma membrane of specific cells involved in the diseaseprocess.

Cellular membranes consist of lipid protein bilayers that are freelypermeable to small, nonionic, lipophilic compounds and inherentlyimpermeable to most natural metabolites and therapeutic agents. Wilson,D. B. Ann. Rev. Biochem. 47:933-965 (1978). The biological and antiviraleffects of natural and modified oligonucleotides in cultured mammaliancells have been well documented, so it appears that these agents canpenetrate membranes to reach their intracellular targets. Uptake ofantisense compounds into a variety of mammalian cells, including HL-60,Syrian Hamster fibroblast, U937, L929, CV-1, and ATH8 cells has beenstudied using natural oligonucleotides and nuclease resistant analogs,such as alkyl triesters, Miller, P. S., Braiterman, L. T. and Ts'O, P.O. P., Biochemistry 16:1988-1996 (1977); methylphosphonates,Marcus-Sekura, C. H., Woerner, A. M., Shinozuka, K. Zon, G., andQuinman, G. V. , Nuc. Acids Res. 15:5749-5763 (1987) and Miller, P. S.,McParland, K. B., Hayerman, K. and Ts'O, P. O. P., Biochemistry20:1874-1880 (1981); and phosphorothioates, Ceruzzi, M. and Draper, K.Nucleosides & Nucleotides 8:815-818 (1989); Miller, P. S., Braiterman,L. T. and Ts'O, P. O. P. Biochemistry 16:1988-1996 (1977) and Loke, S.L., Stein, C., Zhang, X. H. Avigan, M., Cohen, J. and Neckers, L. M.Curr. Top. Microbiol. Immunol. 141:282-289 (1988).

Enhanced cellular uptake has previously been achieved by attachment offunctional groups to the 3' and 5' end of oligonucleotides to enhancecellular uptake in specific cell types. Previous studies have shown thatplasmid DNA complexed with an (asialo)glycoprotein-poly(L-lysine)conjugate, could be targeted to hepatocytes, which contain unique cellsurface receptors for galactose-terminal (asialo)glycoproteins. Wu, G.Y. and Wu, C. H. Biochemistry 27:887-892 (1988). Other groups havesynthesized oligodeoxyribonucleotides that have a 5'-attached alkylatingagent and a 3' attached cholesterol moiety and determined that thesemodified oligonucleotides were taken up into cells more efficiently thancontrol compounds without the steroid moiety. Zon, G. inOligodeoxynucleotides: Antisense Inhibitors of Gene Expression 234-247,ed. J. S. Cohen (CRC Press, Boca Raton Fla., 1989). Letsinger, et al.Proc. Natl. Acad. Sci. U.S.A. 86:653-656 (1989), have also synthesizedcholesteryl-conjugated phosphorothioates whose anti-HIV activity issignificantly greater than natural oligonucleotides with the samesequence. Additional modifications include conjugation ofoligonucleotides to poly(L-lysine) alone. Stevenson, M. and Iversen, P.L. J. Gen. Virol 70:2673-2682 (1989) and Lemaitre, M., Baynard, B. andLeBleu, B. Proc. Natl. Acad. Sci. U.S.A. 84:648-652 (1987). Thismodification enhanced the antiviral activity of the compound studiedpresumably due to increased cellular uptake imparted by the polycationicpoly(L-lysine).

The conjugation of polyamines to oligonucleotides have been found toenhance cellular uptake of oligonucleotides, increased lipophilicity,cause greater cellular retention and increased distribution of thecompound. Vasseur, et al., Nucleosides and Nucleotides, 1991, 10, 107prepared abasic sites at different sites of oligothymidylates by acidhydrolysis. Thereafter the abasic sites were functionalized withfunctionalities such as 3-amino carbazole, 9-amino elipticine andpsoralen. Vasseur, et al. also refers to unpublished results in whichthe functionalities spermidine and proflavin were employed. Le Doan, etal., Nucleic Acids Research 1987, 15, 8643 teaches oligothymidylatescovalently linked to porphyrins at their 3' end via one of the linkers--O----CH₂ --CO--NH--(CH₂)₂ --NH or PO₄ --(CH₂)₆ --NH--. Le Doan, et al.also used the linker PO₄ --(CH₂)₆ --NH-- to link porphyrins to the 5'end of oligothymidylates. Another group, Summerton, et al., U.S. Pat.No. 5,034,506 issued Jul. 23, 1991 teaches morpholino subunits, linkedtogether by uncharged, achiral linkages such as amides. As described inPCT/US91/04086 filed Jun. 10, 1991, polyamines have also been linked atthe 5' end of an oligonucleotide at the 5' site of the sugar moiety ofthe terminal nucleoside and at the 2-position carbon of the heterocyclicbaseof 2'-deoxyadenosine, 2'-deoxyguanosines and other purines andpurine analogs by known procedures as described in PCT/US/91/00243 filedJan. 11, 1991.

Novel amines and methods of preparing the same are greatly needed inorder to enhance cellular uptake of oligonucleotides, increaselipophilicity, cause greater cellular retention and increasedistribution of the compound within the cell. The present inventionfulfills this need.

OBJECTS OF THE INVENTION

It is one object of the present invention to provide novelamine-containing compounds useful in therapeutics.

It is a further object of the present invention to provide methods ofproducing said novel compounds.

It is another object of the present invention to provide methods ofmodulating the production of a protein by an organism.

It is still a further object of the present invention to provide methodsof treating a mammal suffering from a disease characterized by theundesired production of a protein.

It is yet a further object of the present invention to provide methodsof diagnosing the presence of an RNA in a biological sample.

These and other objects will become apparent from the followingdescription and accompanying claims.

SUMMARY OF THE INVENTION

The present invention provides compounds which may have enhancedefficacy as an antisense-based therapy. Compounds of the presentinvention can have enhanced cellular uptake, increased lipophilicity,cause greater cellular retention and demonstrate increased distribution.Furthermore the present invention provides simple methods for synthesisof these novel compounds.

In accordance with some embodiments of the present invention, compoundshaving the structure: ##STR1## wherein R₁ and R₂ are independently H, anucleotide, oligonucleotide, or polyamine and at least one of R₁ and R₂is a purine containing oligonucleotide, R₃ is a linear or cyclicnon-aromatic polyamine species, and X is H, O--R₁₁, S--R₁₁, F, Cl, Br,CN, CF₃, OCF₃, OCN, SOCH₃, SO₂ CH₃, ONO₂, N₃, HN₂, heterocylcoalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,a reporter molecule, an RNA cleaving group, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide whereinR₁₁ is H, C₁ to C₁₀ straight or branched chain lower alkyl orsubstituted lower alkyl, C₂ to C₁₀ straight or branched chain loweralkenyl or substituted lower alkylenyl, C₃ to C₁₀ straight or branchedchain lower alkynyl or substituted lower alkynyl, a ¹⁴ C containinglower alkyl, lower alkenyl or lower alkynyl, C₇ to C₁₄ substituted orunsubstituted alkylaryl or aralkyl, a ¹⁴ C containing C₇ to C₁₄ alkarylor aralkyl, alicyclic, heterocyclic, a reporter molecule, a RNA cleavinggroup, a group for improving the pharmacokinetic properties of anoligonucleotide or a group for improving the pharmacodynamic propertiesof an oligonucleotide, are provided.

In accordance with still other embodiments of the present invention,compounds having the structure: ##STR2## wherein R₄ is anoligonucleotide and M is a pendent group having a polyamine speciesattached thereto are provided.

Methods of preparing such compounds utilizing enzymatic reagents arealso provided in some aspects of the invention. Thus compounds ofFormula I may be prepared by methods comprising the steps of providing asynthon having the structure: ##STR3## wherein R₁ and R₂ areindependently H, a nucleotide, oligonucleotide or polyamine, and atleast one of R₁ and R₂ is a purine containing oligonucleotide, and X isH, O--R₁₁, S--R₁₁, F, Cl, Br, CN, CF₃, OCF₃, OCN, SOCH₃, SO₂ CH₃, ONO₂,N₃, HN₂, heterocylcoalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, a reporter molecule, an RNA cleavinggroup, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide wherein R₁₁ is H, C₁ to C₁₀ straight or branchedchain lower alkyl or substituted lower alkyl, C₂ to C₁₀ straight orbranched chain lower alkenyl or substituted lower alkylenyl, C₃ to C₁₀straight or branched chain lower alkynyl or substituted lower alkynyl, a¹⁴ C containing lower alkyl, lower alkenyl or lower alkynyl, C₇ to C₁₄substituted or unsubstituted alkylaryl or aralkyl, a ¹⁴ C containing C₇to C₁₄ alkaryl or aralkyl, alicyclic, heterocyclic, a reporter molecule,a RNA cleaving group, a group for improving the pharmacokineticproperties of an oligonucleotide or a group for improving thepharmacodynamic properties of an oligonucleotide. Thereafter the synthonis reacted with R₃, wherein R₃ is a linear or cyclic non-aromaticpolyamine species, under reducing conditions to yield the final product.

Compounds of Formula II may also be prepared enzymatically by providinga starting material having the structure: ##STR4## wherein R₄ is anoligonucleotide, R₁₂ is an oligonucleotide and B is urea or aheterocyclic base having a corresponding glycosylase and reacting thestarting material with an endonuclease to generate a conjugatedα,β-unsaturated system in the sugar residue of the 3' terminalnucleotide. Thereafter the compound having a conjugated α,β-unsaturatedsystem is reacted with a pendent group containing a nucleophilefunctionality thereon. Following addition of the pendent group thedouble bond of the α,β system is reduced with a reducing agent. Apolyamine species may then be attached to the pendent group via analkylation reaction. Alternatively, a polyamine species may be attachedto a pendent group which is a bifunctional linker.

In accordance with still other embodiments of the present inventioncompounds having the structure: ##STR5## wherein R₄ is anoligonucleotide, R₅ is a linear or cyclic non-aromatic polyamine speciescontaining at least one non-amide nitrogen atom, and R₆ is H, a purineheterocycle or a pyrimidine heterocycle, are provided. Methods ofpreparing compounds of Formula III are also provided in some aspects ofthe present invention comprising the steps of reacting anoligonucleotide having a 3' ribofuranosyl sugar with an oxidizing agentto produce an activated dialdehyde-terminated oligonucleotide andreacting said activated oligonucleotide with a linear or cyclicnon-aromatic polyamine species under reducing conditions to yield saidcompound.

In accordance with other aspects of the invention compounds having thestructure: ##STR6## wherein B is a purine or pyrimidine heterocyclicbase, R₈ and R₉ are independently H, PO₂, a nucleotide, oligonucleotideor polyamine species and at least one of R₈ and R₉ is a purinecontaining oligonucleotide, and at least one of R₈, R₉ and A is aspecies comprising the formula L₁ -L₂ -polyamine wherein L₁ is an aminolinker and L₂ is a heterobifunctional linker; and wherein if R₈ is not apurine containing oligonucleotide or polyamine species, then R₈ is anucleotide or PO₂ ; if R₉ is not a purine containing oligonucleotide orpolyamine species, then R₉ is H or a nucleotide; and if A is not apolyamine species then A is H or OH are provided.

Therapeutic and diagnostic methods are also encompassed by the presentinvention. Methods of modulating the production of protein by anorganism comprising contacting an organism with a compound having thestructure of Formula I, Formula II, Formula III or Formula IV areencompassed by some embodiments of the present invention. In otheraspects of the invention, methods of treating an animal having a diseasecharacterized by undesired production of protein comprising contactingan animal with a compound having the structure of Formula I, Formula II,Formula III, or Formula IV in a pharmaceutically acceptable carrier areprovided. Still other methods of the present invention provide methodsfor detecting the presence or absence of an RNA in a biological samplesuspected of containing said RNA are provided comprising contacting asample with a compound having the structure of Formula I, Formula II,Formula III or Formula IV wherein the compound is specificallyhybridizable with the RNA and detecting the presence or absence ofhybridization of the compound to the sample wherein hybridization isindicative of the presence of RNA in the sample.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of one preferred syntheses ofcompounds of Formula I.

FIG. 2 is a schematic representation of one preferred syntheses ofcompounds of Formula II.

FIG. 3 is a schematic representation of one preferred syntheses ofcompounds of Formula III.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides novel amine compounds useful forantisense therapy. In one embodiment of the present invention compoundshaving the structure: ##STR7## wherein R₁ and R₂ are independently H, anucleotide, oligonucleotide, or polyamine and at least one of R₁ and R₂is a purine containing oligonucleotide, R₃ is a linear or cyclicnon-aromatic polyamine species, and X is H, O--R₁₁, S--R₁₁, F, Cl, Br,CN, CF₃, OCF₃, OCN, SOCH₃, SO₂ CH₃, ONO₂, N₃, HN₂, heterocylcoalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,a reporter molecule, an RNA cleaving group, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide whereinR₁₁ is H, C₁ to C₁₀ straight or branched chain lower alkyl orsubstituted lower alkyl, C₂ to C₁₀ straight or branched chain loweralkenyl or substituted lower alkylenyl, C₃ to C₁₀ straight or branchedchain lower alkynyl or substituted lower alkynyl, a ¹⁴ C containinglower alkyl, lower alkenyl or lower alkynyl, C₇ to C₁₄ substituted orunsubstituted alkylaryl or aralkyl, a ¹⁴ C containing C₇ to C₁₄ alkarylor aralkyl, alicyclic, heterocyclic, a reporter molecule, a RNA cleavinggroup, a group for improving the pharmacokinetic properties of anoligonucleotide or a group for improving the pharmacodynamic propertiesof an oligonucleotide, are provided. In some embodiments of the presentinvention both R₁ and R₂ are oligonucleotides, at least one of whichincludes at least one purine nucleotide.

In the context of this invention, the term "oligonucleotide" refers to apolynucleotide formed from naturally occurring bases, such as purine andpyrimidine heterocycles, and furanyl groups joined by nativephosphodiester bonds. This term effectively refers to naturallyoccurring species or synthetic species formed from naturally occurringsubunits or their close homologs. The term "oligonucleotide" may alsorefer to moieties which have portions similar to naturally occurringoligonucleotides but which have non-naturally occurring portions. Thus,oligonucleotides may have altered sugar moieties or inter-sugarlinkages. Exemplary among these are the phosphorothioate and othersulfur-containing species which are known for use in the art. Inaccordance with some preferred embodiments, at least some of thephosphodiester bonds of the oligonucleotide have been substituted with astructure which functions to enhance the stability of theoligonucleotide or the ability of the oligonucleotide to penetrate intothe region of cells where the viral RNA is located. It is preferred thatsuch substitutions comprise phosphorothioate bonds, phosphotriesters,methyl phosphonate bonds, short chain alkyl or cycloalkyl structures orshort chain heteroatomic or heterocyclic structures. Most preferred areCH₂ --NH--O--CH₂, CH₂ --N(CH₃)--O--CH₂, CH₂ --O--N(CH₃)--CH₂, CH₂--N(CH₃)--N(CH₃)--CH₂ and O--N(CH₃)--CH₂ --CH₂ structures wherephosphodiester is O--P--O--CH₂). Also preferred are morpholinostructures. Summerton, J. E. and Weller, D. D., U.S. Pat. No. 5,034,506issued Jul. 23, 1991. In other preferred embodiments, such as theprotein-nucleic acid (PNA) backbone, the phosphodiester backbone of theoligonucleotide may be replace with a polyamide backbone, the basesbeing bound directly or indirectly to the aza nitrogen atoms of thepolyamide backbone. P. E. Nielsen, et al., Science 1991 254 1497. Inaccordance with other preferred embodiments, the phosphodiester bondsare substituted with other structures which are, at once, substantiallynon-ionic and non-chiral, or with structures which are chiral andenantiomerically specific. Still other linkages include the thosedisclosed in U.S. patent applications Ser. No. 566,836, filed Aug. 13,1990, entitled Novel Nucleoside Analogs; Ser. No. 703,619, filed May 21,1991, entitled Backbone Modified Oligonucleotide Analogs; Ser. No.903,160, filed Jun. 24, 1992, entitled Heteroatomic OligonucleosideLinkages; Ser. No. PCT/US92/04294, filed May 21, 1992, entitled BackboneModified Oligonucleotides; and Ser. No. PCT/US92/04305, all assigned tothe assignee of this invention. Persons of ordinary skill in the artwill be able to select other linkages for use in practice of theinvention.

Oligonucleotides may also include species which include at least somemodified base forms. Thus, purines and pyrimidines other than thosenormally found in nature may be so employed. For example, deaza or azapurines and pyrimidines may be used in place of naturally purine orpyrimidine bases and pyrimidine bases having substituent groups at the5- or 6-positions; purine bases having altered or replacementsubstituent groups at the 2-, 6- or 8-positions are also provided insome aspects of the present invention. Similarly, modifications on thefuranosyl portion of the nucleotide subunits may also be effected, aslong as the essential tenets of this invention are adhered to. Examplesof such modifications are 2'--O-alkyl- and 2'-halogen-substitutednucleotides. Some specific examples of modifications at the 2' positionof sugar moieties which are useful in the present invention are OH, SH,SCH₃, F, OCN, O(CH₂)_(n) NH₂, O(CH₂)_(n) CH₃ where n is from 1 to about10; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl;Cl, Br, CN, CF₃, OCF₃, O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH₃,SO₂ CH₃ ; ONO₂ ; NO₂ ; N₃ ; NH₂ ; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleavinggroup; a conjugate; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. Sugar mimetics such ascyclobutyls may also be used in place of the pentofuranosyl group.Exemplary modifications are disclosed in U.S. patent applications: Ser.No. 463,358, filed Jan. 11, 1990, entitled Compositions And Methods ForDetecting And Modulating RNA Activity; Ser. No. 566,977, filed Aug. 13,1990, entitled Sugar Modified Oligonucleotides That Detect And ModulateGene Expression; Ser. No. 558,663, filed Jul. 27, 1990, entitled NovelPolyamine Conjugated Oligonucleotides; Ser. No. 558,806, filed Jul. 27,1991, entitled Nuclease Resistant Pyrimidine Modified OligonucleotidesThat Detect And Modulate Gene Expression; and Ser. No. PCT/US91/00243,filed Jan. 11, 1991, entitled Compositions and Methods For Detecting AndModulating RNA Activity; Ser. No. 777,670, filed Oct. 15, 1991, entitledOligonucleotides Having Chiral Phosphorus Linkages; Ser. No. 814,961,filed Dec. 24, 1991, entitled Gapped 2' Modified PhosphorothioateOligonucleotides; Ser. No. 808,201, filed Dec. 13, 1991, entitledCyclobutyl Oligonucleotide Analogs; and Ser. No. 782,374, filed 782,374,entitled Derivatized Oligonucleotides Having Improved Uptake & OtherProperties, all assigned to the assignee of this invention. Thedisclosures of all of the above noted patent applications areincorporated herein by reference. Oligonucleotides may also compriseother modifications consistent with the spirit of this invention. Sucholigonucleotides are best described as being functionallyinterchangeable with yet structurally distinct from naturaloligonucleotides. All such oligonucleotides are comprehended by thisinvention so long as they effectively function as subunits in theoligonucleotide. Thus, purine containing oligonucleotide areoligonucleotides comprising at least one purine base or analog thereof.In other embodiments of the present invention compounds of the presentinvention may be "subunits" of a species comprising two or morecompounds of the present invention which together form a singleoligonucleotide.

Oligonucleotides of the present invention may be naturally occurring orsynthetically produced and may range in length from about 8 to about 50nucleotides. In more preferred embodiments of the present invention saidoligonucleotides may be from 8 to 40 nucleotides in length. Mostpreferably, oligonucleotides of the present invention may be from 12 toabout 20 nucleotides in length.

The phrase polyamine species as used throughout the specification refersto species that have a plurality of nitrogen atoms thereon. Polyaminesinclude primary amines, hydrazines, semicarbazines, thiosemicarbazinesand similar nitrogenous species. Such species can be symmetrical speciessuch as polyamine containing polymers or they can be unsymmetricalwherein the amine functionalities of the polyamine are separated inspace by different moieties. In addition to carbon atoms other atomicspecies such as nitrogen and sulfur may also be incorporated into thepolyamine species. In some preferred embodiments of the invention, atleast one nitrogen atom of the polyamine has a free electron pair.

Preferred as polyamine species are species that range in length fromabout 3 to about 20 units. More preferably species having at least onenitrogen atom have the general formula H₂ N (CH₂)_(n) NH!_(m) -- whereinn is an integer between 2 and 8 and m is an integer between 1 and 10.These species can be linear or cyclic. Cyclic amines would include crownamines and mixed crown amines/crown ethers.

Other suitable nitrogen-containing compound suitable for the formationof polyamine species include C₁ -C₂₀ straight chain alkylamine, C₁ -C₂₀straight chain substituted alkylamine, C₂ -C₅₀ branched chainalkylamine, C₂ -C₅₀ branched chain substituted alkylamine, C₃ -C₅₀cyclic alkylamine, C₃ -C₅₀ cyclic substituted alkylamine, C₂ -C₂₀straight chain alkenylamine, C₂ -C₂₀ straight chain substitutedalkenylamine, C₃ -C₅₀ branched chain alkenylamine, C₃ -C₅₀ branchedchain substituted alkenylamine, C₃ -C₅₀ cyclic alkenylamine, C₃ -C₅₀cyclic substituted alkenylamine, C₂ -C₂₀ straight chain alkynylamine, C₂-C₂₀ straight chain substituted alkynylamine, C₃ -C₅₀ branched chainalkynylamine, C₃ -C₅₀ branched chain substituted alkynylamine, C₃ -C₅₀cyclic alkynylamine, C₃ -C₅₀ cyclic substituted alkynylamine, C₁ -C₂₀straight chain alkylhydrazine, C₁ -C₅₀ straight chain substitutedalkylhydrazine, C₂ -C₅₀ branched chain alkylhydrazine, C₂ -C₅₀ branchedchain substituted alkylhydrazine, ₃ -C₅₀ cyclic hydrazoalkane, C₃ -C₅₀cyclic substituted hydrazoalkane, C₂ -C₂₀ straight chainalkenylhydrazine, C₂ -C₂₀ straight chain substituted alkenylhydrazine,C₃ -C₅₀ branched chain alkenylhydrazine, C₃ -C₅₀ branched chainsubstituted alkenylhydrazine, C₃ -C₅₀ cyclic hydrazoalkene, C₃ -C₅₀cyclic substituted hydrazoalkene,C₂ -C₂₀ straight chainalkynylhydrazine, C₂ -C₂₀ straight chain substituted alkynylhydrazine,C₃ -C₅₀ branched chain alkynylhydrazine, C₃ -C₅₀ branched chainsubstituted alkynylhydrazine, C₃ -C₅₀ cyclic hydrazoalkyne, C₃ -C₅₀cyclic substituted hydrazoalkyne, C₁ -C₂₀ straight chainalkylhydroxyamine, C₁ -C₂₀ straight chain substituted alkylhydroxyamine,C₂ -C₅₀ branched chain alkylhydroxyamine, C₂ -C₅₀ branched chainsubstituted alkylhydroxyamine, C₃ -C₅₀ cyclic oxyalkylamine, C₃ -C₅₀cyclic substituted oxyalkylamine, C₂ -C₂₀ straight chainalkenylhydroxyamine, C₂ -C₂₀ straight chain substitutedalkenylhydroxyamine, C₃ -C₅₀ branched chain alkenylhydroxyamine, C₃ -C₅₀branched chain substituted alkenylhydroxyamine, C₃ -C₅₀ cyclicoxyalkenylamine, C₃ -C₅₀ cyclic substituted oxyalkenylamine, C₂ -C₂₀straight chain alkynylhydroxyamine, C₂ -C₂₀ straight chain substitutedalkynylhydroxyamine, C₃ -C₅₀ branched chain alkynylhydroxyamine, C₃ -C₅₀branched chain substituted alkynylhydroxyamine, C₃ -C₅₀ cyclicoxyalkynylamine, C₃ -C₅₀ cyclic substituted oxyalkynylamine, C₁ -C₂₀straight chain alkylsemicarbazide, C₁ -C₂₀ straight chain substitutedalkylsemicarbazide, C₂ -C₅₀ branched chain alkylsemicarbazide, C₂ -C₅₀branched chain substituted alkylsemicarbazide, C₃ -C₅₀ cyclicalkylsemicarbazide, C₃ -C₅₀ cyclic substituted alkylsemicarbazide, C₂-C₂₀ straight chain alkenylsemicarbazide, C₂ -C₂₀ straight chainsubstituted alkenylsemicarbazide, C₃ -C₅₀ branched chainalkenylsemicarbazide, C₃ -C₅₀ branched chain substitutedalkenylsemicarbazide, C₃ -C₅₀ cyclic alkenylsemicarbazide, C₃ -C₅₀cyclic substituted alkenylsemicarbazide, C₂ -C₂₀ straight chainalkynylsemicarbazide, C₂ -C₂₀ straight chain substitutedalkynylsemicarbazide, C₃ -C₅₀ branched chain alkynylsemicarbazide, C₃-C₅₀ branched chain substituted alkynylsemicarbazide, C₃ -C₅₀cyclicalkynylsemicarbazide, C₃ -C₅₀ cyclic substitutedalkynylsemicarbazide, C₁ -C₂₀ straight chain alkylthiosemicarbazide, C₁-C₂₀ straight chain substituted alkylthiosemicarbazide, C₂ -C₅₀ branchedchain alkylthiosemicarbazide, C₂ -C₅₀ branched chain substitutedalkylthiosemicarbazide, C₃ -C₅₀ cyclic alkylthiosemicarbazide, C₃ -C₅₀cyclic substituted alkylthiosemicarbazide, C₂ -C₂₀ straight chainalkenylthiosemicarbazide, C₂ -C₂₀ straight chain substitutedalkenylthiosemicarbazide, C₃ -C₅₀ branched chainalkenylthiosemicarbazide, C₃ -C₅₀ branched chain substitutedalkenylthiosemicarbazide, C₃ -C₅₀ cyclic alkenylthiosemicarbazide, C₃-C₅₀ cyclic substituted alkenylthiosemicarbazide, C₂ -C₂₀ straight chainalkynylthiosemicarbazide, C₂ -C₂₀ straight chain substitutedalkynylthiosemicarbazide, C₃ -C₅₀ branched chainalkynylthiosemicarbazide, C₃ -C₅₀ branched chain substitutedalkynylthiosemicarbazide, C₃ -C₅₀ cyclic alkynylthiosemicarbazide, C₃-C₅₀ cyclic substituted alkynylthiosemicarbazide, C₁ -C₂₀ straight chainalkylhydrazone, C₁ -C₂₀ straight chain substituted alkylhydrazone, C₂-C₅₀ branched chain alkylhydrazone, C₂ -C₅₀ branched chain substitutedalkylhydrazone, C₃ -C₅₀ cyclic hydrazoalkane, C₃ -C₅₀ cyclic substitutedhydrazoalkane, C₂ -C₂₀ straight chain alkenylhydrazone, C₂ -C₂₀ straightchain substituted alkenylhydrazone, C₃ -C₅₀ branched chainalkenylhydrazone, C₃ -C₅₀ branched chain substituted alkenylhydrazone,C₃ -C₅₀ cyclic hydrazoalkene, C₃ -C₅₀ cyclic substituted hydrazoalkene,C₂ -C₂₀ straight chain alkynylhydrazone, C₂ -C₂₀ straight chainsubstituted alkynylhydrazone, C₃ -C₅₀ branched chain alkynylhydrazone,C₃ -C₅₀ branched chain substituted alkynylhydrazone, C₃ -C₅₀ cyclichydrazoalkyne, C₃ -C₅₀ cyclic substituted hydrazoalkyne, C₁ -C₂₀straight chain alkylhydrazide, C₁ -C₂₀ straight chain substitutedalkylhydrazide, C₃ -C₅₀ branched chain alkylhydrazide, C₃ -C₅₀ branchedchain substituted alkylhydrazide, C₃ -C₅₀ cyclic alkylhydrazide, C₃ -C₅₀cyclic substituted alkylhydrazide, C₂ -C₂₀ straight chainalkenylhydrazide, C₂ -C₂₀ straight chain substituted alkenylhydrazide,C₃ -C₅₀ branched chain alkenylhydrazide, C₃ -C₅₀ branched chainsubstituted alkenylhydrazide, C₃ -C₅₀ cyclic alkenylhydrazide, C₃ -C₅₀cyclic substituted alkenylhydrazide, C₂ -C₂₀ straight chainalkynylhydrazide, C₂ -C₂₀ straight chain substituted alkynylhydrazide,C₃ -C₅₀ branched chain alkynylhydrazide, C₃ -C₅₀ branched chainsubstituted alkynylhydrazide, C₃ -C₅₀ cyclic alkynylhydrazide and C₃-C₅₀ cyclic substituted alkynylhydrazide.

In accordance with preferred embodiments of the present inventionpolyamine species are linear or cyclic non-aromatic polyamine species.In still more preferred embodiments of the present invention polyaminespecies are linear or cyclic non-aromatic comprising non-amide nitrogenatoms. By non-amide is meant a nitrogen which is not adjacent to acarbonyl group (i.e. C═O or C═S).

In still other embodiments of the present invention compounds having thestructure: ##STR8## wherein R₄ is an oligonucleotide and M is a pendentgroup having a polyamine species attached thereto are provided. Thependent group may be any group to which a polyamine may be attached. Inpreferred embodiments the pendent group is a R₁₀ S or R₁₀ NH, whereinR₁₀ is any of a broad range of reactive groups effective for subsequentattachment of polyamine species to the pendent group. Suitable for R₁₀are substituted and un-substituted, straight chain or branched chainedC₁ -C₂₀ alkyl groups or substituted or un-substituted C₇ -C₁₄ arylgroups having the nucleophile in one position thereon and a furtherfunctional group in a further position thereon. The pendent group maythus, subsequently functionalized with a bifunctional linker groupamendable for attachment of a polyamine species to the pendent group.Alternatively the polyamine species may be directly attached to apendent group such as by alkylation.

Further in accordance with the present invention are provided compoundshaving the structure: ##STR9## wherein R₄ is an oligonucleotide, R₅ is alinear or cyclic non-aromatic polyamine species containing non-amidenitrogen atoms, and R₆ is H, a purine heterocycle or a pyrimidineheterocycle.

The present invention also provides novel polyamine containing compoundshaving the structure: ##STR10## wherein B is a purine or pyrimidineheterocycle, R₈ and R₉ are independently H, PO₂ ⁻, a nucleotide,oligonucleotide or polyamine species and at least one of R₈ and R₉ is apurine containing oligonucleotide, and at least one of R₈, R₉ and A is aspecies comprising the formula L₁ -L₂ -polyamine wherein L₁ is an aminolinker and L₂ is a heterobifunctional linker; and wherein if R₈ is not apurine containing oligonucleotide or polyamine species, then R₈ is anucleotide or PO₂ ⁻ ; if R₉ is not a purine containing oligonucleotideor polyamine species, then R₉ is H or a nucleotide; and if A is not apolyamine species then A is H or OH.

Thus R₈ and R₉ may be oligonucleotides and A may be a species comprisingthe formula L₁ -L₂ -polyamine, or alternatively, R₈ may be anoligonucleotide and one or both of R₉ and A may be a species comprisingthe formula L₁ -L₂ -polyamine; or R₉ may be an oligonucleotide and oneor both of R₈ and A may be a species comprising the formula L₁ -L₂-polyamine. Furthermore, when R₈ is not a purine containingoligonucleotide or polyamine species, then R₈ is a nucleotide or PO₂ ⁻.If R₉ is not a purine containing oligonucleotide or polyamine species,then R₉ is H or a nucleotide, and if A is not a polyamine species then Ais H or OH.

In preferred embodiments of the present invention commercially availableamino linkers may be used. For example, the 3' amino modifiers havingthe trade names C3 CPG and C7 CPG available through Glen Research may beemployed. 5' amino modifiers may also be used such as C3 and C7 5'branched modifiers available through Glen Research. Similarly, 2' aminomodifiers are also envisioned for use in some aspects of the presentinvention. The amino linkers are designed to functionalize a targetoligonucleotide by the introduction of a primary amine at a designatedsite, be it 2', 3' or 5'. As will be apparent to one skilled in the art,any linker which meets this end is encompassed by the present invention.

Likewise, bifunctional linkers effective for purposes of the presentinvention are available commercially. For example,bis-(maleimido)-methyl ether (BMME), disuccinimidyl suberate (DSS),3-maleimidobenzoyl-N-hydroxy-succinimide (MBS),maleimidohexanoyl-N-hydroxyl-succinimide (MHS) andN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) may be useful insome embodiments of the present invention. Other useful bifunctionallinkers will be apparent to one skilled in the art as for instance fromPierce, Rockford, Ill.

Compounds of the present invention may be prepared by providing anoligonucleotide comprising one or more abasic sites. In the context ofthe present invention "abasic site" refers to a nucleotide unit in whichthe purine or pyrimidine group has been removed or replaced by anon-naturally occurring group such as a hydroxyl group. One or moreabasic sites may be incorporated into one or more nucleotide bases of anoligonucleotide to form a synthon having the structure: ##STR11##wherein R₁ and R₂ are independently H, a nucleotide, oligonucleotide orpolyamine species and at least one of R₁ and R₂ is a purine containingoligonucleotide, and X is H, O--R₁₁, S--R₁₁, F, Cl, Br, CN, CF₃, OCF₃,OCN, SOCH₃, SO₂ CH₃,ONO₂, N₃, HN₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, a reporter molecule,an RNA cleaving group, a group for improving the pharmacokineticproperties of an oligonucleotide, or a group for improving thepharmacodynamic properties of an oligonucleotide wherein R₁₁ is H, C₁ toC₁₀ straight or branched chain lower alkyl or substituted lower alkyl,C₂ to C₁₀ straight or branched chain lower alkenyl or substituted loweralkylenyl, C₃ to C₁₀ straight or branched chain lower alkynyl orsubstituted lower alkynyl, a ¹⁴ C containing lower alkyl, lower alkenylor lower alkynyl, C₇ to C₁₄ substituted or unsubstituted alkylaryl oraralkyl, a ¹⁴ C containing C₇ to C₁₄ alkaryl or aralkyl, alicyclic,heterocyclic, a reporter molecule, a RNA cleaving group, a group forimproving the pharmacokinetic properties of an oligonucleotide or agroup for improving the pharmacodynamic properties of anoligonucleotide.

An enzymatic process may be used to produce such a synthon having abasicsites by reaction of a DNA glycosylase with an oligonucleotide startingmaterial. For example, uracil DNA glycosylase act on uracil bases withinan oligonucleotide to create abasic sites. Of course it should berecognized that enzymatic methods using DNA glycosylase may be lesseffective for oligonucleotides more closely resembling RNA such asoligonucleotides having 2' modifications.

Enzymes, as employed in the present invention, may be derived fromnaturally occurring sources or may be prepared by recombinanttechniques. Many useful enzymes are available commercially.

Such synthons may alternatively be prepared by incorporation of abasicsites into an oligonucleotide via abasic sugar precursors. For example,5-O-(4,4'-dimethoxytrityl)-1,2-dideoxy-1-(o-nitrobenzoyl)-D-ribofuranose-3-O-(2-Cyanoethyl-N,N'-diisopropyl)phosphoramidite may be prepared by modification of the procedures ofLyer, et al., Nucleic Acids Research 18: 2855 (1990) and Didier, et al.,Tetrahedron Letters 32: 207 (1991).

Phosphoramidites having a 2' substitutions and abasic sites may also beprepared. For example, a synthon may have 2'-O-methyl or 2'-fluorosubstitutions. Such phosphoramidite may be incorporated into anoligonucleotide by standard procedures. An o-nitrobenzyldeoxyfuranosecontaining oligonucleotide can be synthesized in accordance with theseprocedures. Post synthesis photolysis utilizing a high intensity Hg lampgenerates the corresponding abasic site-containing polymer. In addition,other methods of introducing abasic sites at the 3', 5' and internalpositions of an oligonucleotide to form a synthon are known to thoseskilled in the art. Thereafter the synthon may be reacted with apolyamine species under reducing conditions. As illustrated in FIG. 1,Step A, a compound may be prepared wherein B is uridine and an enzymaticprocess may be used to produce a synthon having abasic sites at one ormore uridine sites by digestion of the compound with an enzyme such asuracil-DNA glycosylase. Other glycosylases will be effective fordifferent targets. As described above, a glycosylase may be determinedby the combined sequence of R₁, R₂ and B. Some useful glycosylases andtheir respective targets are described for example, by Friedberg, DNARepair (W. H. Freeman and Company, NY, 1985) p. 153, incorporated byreference herein in its entirety. These enzymes are commerciallyavailable or may be prepared from known procedures in the art.

In other embodiments of the present invention as exemplified in FIG. 2compound of Formula II may be prepared by providing starting materialhaving the structure: ##STR12## wherein R₄ is an oligonucleotide, R₁₂ isan oligonucleotide and B is urea or a heterocyclic base having acorresponding glycosylase and reacting the compound with an endonucleaseto produce the compound 2 as described by Manoharan, et al., JACS, 1988,110, 2690. Thereafter, the compound 2 is contacted with a pendent groupsuch as R₁₀ S, and reduced with the reducing agent NaCNBH₄ to stabilizethe product 5. A polyamine species may then be added such as byalkylation to provide the final product 7. A polyamine species mayalternatively be added directly to a bifunctional pendent group. Someendonucleases which will be useful in embodiments of the presentinvention are described, for example, in Doetsch and Cunningham,Mutation Research, 1990, 236, 173, incorporated by reference herein inits entirety. The endonuclease chosen will depend upon the identity of Band the sequence of R₄ and/or R₁₂. Thus, if B is a pyrimidineheterocycle, and the sequence of R₁₂ begins with a pyrimidine, then anendonuclease such as T4 or M. luteus UV endonuclease may be chosen.Following digestion by T4 or M. luteus UV endonuclease, B and R₁₂ areremoved, resulting in a 3' terminal α,β unsaturated aldehydic species.In some instances, it may be desireable to engineer the sequence of thespecies so as to provide a endonuclease digestion site at a desiredlocation.

Thus, in one preferred embodiment of the present invention R₄ may beTGGGAGCCATAGCGAGGC (SEQ ID NO: 1), B may be the pyrimidine thymine andR₁₂ may be a thymidine dinucleotide. The net result of digestion of thisspecies with T4 UV endonuclease will be TGGGAGCCATAGCGAGGCN (SEQ ID NO:2) wherein N represents the aldehydic species.

Treatment of the digested compound with pendent group comprising alinker bearing a nucleophile results in the addition of the pendentgroup at the 3' terminus of the compound to join the linker to thedigested compound. Suitable nucleophilic species include thiols andamines moieties as described above. In preferred embodiments of thepresent invention the pendent group is R₁₀ S⁻ or R₁₀ NH. A polyaminespecies such as NH₂ (CH₂)_(n) NH₂ wherein n is an integer from 1 toabout 10 could be used as the attacking nucleophile by suitably blockingone end thereof and utilizing the other end as the attackingnucleophilic species. R₁₀ can be further selected to provide a linkageor bridge between the nucleophile and a polyamine. Suitable for R₁₀ aresubstituted and un-substituted, straight chain or branched chained C₁-C₂₀ alkyl groups or substituted or un-substituted C₇ -C₁₄ aryl groupshaving the nucleophile in one position thereon and a further functionalgroup in a further position thereon. After attachment of the pendentgroup via nucleophilic attack on compound 2, for attachment of thepolyamine species the further functional group is then derivitizedeither via a bifunctional linking group, an alkylation type reaction orother derivation reaction known to those skilled in the art.

Upon addition of the pendent group to the digested compound, the doublebond remaining on the digested compound is reduced to stabilize theproduct. Reducing agents effective to stabilize the end product of sucha reaction are well known in the art. Some suitable reducing agentsinclude sodium cyanoborohydride, lithium cyanoborohydride and sodiumborohydride.

Thereafter a polyamine may be added via an alkylation reaction ordirectly to a pendent group which is a bifunctional linker. The compoundmay further be derivatized by attaching one or more reactive groups toat least one of the nitrogen atoms of the polyamine species. Reactivegroups include, but are not limited to reporter groups, alkylatingagents, intercalating agents, RNA cleaving moieties, cell receptorbinding molecules, steroids, peptides, crown amines, porphyrins andcross-linking agents.

In accordance with other methods of the present invention compounds ofFormula III may be prepared by reacting an oligonucleotide having a 3'ribofuranosyl sugar with an oxidizing agent to produce andialdehyde-terminated activated oligonucleotide. Suitable oxidantsinclude periodate solution, lead tetraacetate, activated MnO₂, thallium(III) salts, pyridinium chlorochromate and O₂ catalyzed by Co (III)salts.

Thereafter the dialdehyde-terminated activated oligonucleotide isreacted with a polyamine species under reducing conditions. Reducingagents are known to those skilled in the art. Preferably, the activatedoligonucleotide and species containing at least one nitrogen atom willbe reacted in the presence of a solution of sodium cyanoborohydride,lithium cyanoborohydride or sodium borohydride.

In preferred embodiments of the present invention compounds may beproduced as illustrated by FIG. 3, by preparation of an oligonucleotidehaving a 3' ribofuranosyl end followed by attack of the 3' ribofuranosylring by an oxidant such as m-periodate solution in 0.1M NaOac bufferpH5, as described by Bayard, Bisbal and Lebleu in Biochemistry 25: 3730(1986) to produce a dialdehyde-terminated activated oligonucleotide(FIG. 3, Step A). The activated oligonucleotide and a species containingfour nitrogen atoms, spermine, can be reacted in the presence of thereducing agent, sodium cyanoborohydride (FIG. 3, Step B).

Compounds of the present invention are preferably specificallyhydridizable with a target region. By "specifically hybridizable" hereinis meant capable of forming a stable duplex with a target DNA or RNA. Itis believed that oligonucleotides which form Watson-Crick base pairs,i.e. are complementary with target DNA or RNA and which specificallyhybridize with target DNA or RNA inhibit the flow of genetic informationfrom DNA to protein. In some embodiments of the present invention theoligonucleotide portions of compounds of the present invention are atleast 70% complementary to a target sequence. In preferred embodimentsof the present invention the oligonucleotide portions of compounds ofthe present invention are at least 80% complementary to a targetsequence. 100% complementarity of the oligonucleotide portions ofcompounds of the present invention to a target sequence is mostpreferred. In preferred embodiments of the present invention, theoligonucleotide portions may be specifically hybridizable with DNA orRNA from papilloma virus, herpes viruses, human immunodeficiency virus,Candida, cytomegaloviruses, and influenza viruses. In addition, theoligonucleotide portions may also be specifically hybridizable withendogenous DNA or RNA of a cell. By oligonucleotide portions is meant R₁and/or R₂ of Formula I, R₄ of Formula II, R₄ and/or R₆ of Formula III,or R₈ and/or R₉ of Formula IV.

For therapeutics, an animal suspected of having a disease characterizedby excessive or abnormal production of a protein is treated byadministering a compound having the structure set forth in Formula I,Formula II, Formula III, or Formula IV in a pharmaceutically acceptablecarrier. Most preferable, the compound is hybridizable with an RNAcoding for the protein. Persons of ordinary skill in the art can easilydetermine optimum dosages, dosing methodologies and repetition rates.Such treatment is generally continued until either a cure is effected ora diminution in the diseased state is achieved. Long term treatment islikely for some diseases.

The compounds of the present invention will also be useful as a researchreagent useful for the modulation of the production of a protein by anorganism. Modulation may be accomplished by contacting the organism withcompounds of the present invention having structures as set forth inFormula I, Formula II, Formula III, or Formula IV. Preferably thecompounds are hybridizable with RNA coding for the protein.

Diagnostic applications include the detection of the presence or absenceof an RNA in a sample suspected of containing RNA comprising contactingthe sample with a compound having structures as set forth in Formula I,Formula II, Formula III or Formula IV wherein the compound isspecifically hybridizable with the RNA and detecting the presence orabsence of hybridization of the compound to the sample whereinhybridization is indicative of the present of the RNA in the sample.

It is also envisioned by the present invention to provide compounds inwhich at least one of the nitrogen atoms of the polyamine arederivatized with one or more of the group consisting of functionalitiessuch as reporter groups, alkylating agents, intercalating agents, cellreceptor binding molecules, steroids, crown amines, porphyrins andcross-linking agents. Therapeutic, diagnostic and research reagentapplications are equally, or even more effective when the polyaminespecies further comprises such groups. Such compounds allow greaternumbers of functionalities to be delivered to a target. For example,reporter groups such as biotin, fluorescent molecules and variousfluorophores may be attached to compounds of the present invention toeffect diagnostic ends, resulting in signal amplification as compared toconventional oligonucleotide-reporter group combinations. In a preferredembodiment of the present invention, biotin may be used to functionalizecompounds of the present invention by reacting a compound withD-biotin-N-hydroxysuccinimide ester. In a further preferred embodiment,the polyamine species may be further functionalized by reacting thecompound containing the polyamine species with an activated ester havingthe structure: ##STR13## to form a compound with repeating imidazolecatalytic cleaver units useful as an antisense therapeutic agents.Heterobifunctional linkers also can be utilized for attachment ofintercalators, RNA cleaving agents including imidazoles, cell receptorbinding molecules, steroids, alkylating agents, crown amines, porphyrinsand cross-linkers to the polyamine species.

The following examples are illustrative but are not meant to be limitingof the present invention.

EXAMPLES Example 1

Preparation of an Abasic Site Containing Oligonucleotide via EnzymaticReaction

A. Synthesis of an Oligonucleotide Containing a Single Uridine Site

An oligonucleotide having the sequence CGC AGU CAG CC (SEQ ID NO:3)wherein U represents a 2' deoxyuridine nucleotide, was prepared bystandard solid phase synthesis. The deoxyuridine nucleotide in themiddle of the sequence was added during synthesis utilizing deoxyuridinephosphoramidite (Glen Research, Sterling, Va.). The oligonucleotide wasprepared utilizing standard synthesis cycles. It was deprotected bynormal deprotection at 55° C. utilizing ammonium hydroxide, 30%, for 16hours. The solvent was evaporated and the residue was purified by HPLCand detritylated. Final purification was effected on Sephadex G-25.

B. Preparation of Enzyme Stock Solution

Uracil-DNA glycosylase was isolated from E. Coli M5219 cells transformedwith the expression plasmid pBD396 containing the ung gene. The enzymewas purified by electrophoretic homogeneity as described by Lindahl etal., J. Biol. Chem. 252: 3286 (1977) and stored in 30 mM HEPES-NaOH, pH7.4, containing 5% glycerol, 2 mM DTT and 1 mM EDTA.

C. Preparation of Oligonucleotide Containing Single Abasic Site

An abasic oligonucleotide of the sequence CGC AGN CAG CC (SEQ ID NO:4)wherein N represents an abasic site, was prepared by treating 237 O.D.units of an oligonucleotide having SEQ ID NO:1 of Example 1A in 0.5 mlwater with 200 μl of the stock solution of Example 1B (200 μg of uracilDNA-glycosylase) and incubating at room temperature overnight. HPLCanalysis showed quantitative removal of uracil as indicated by a 1:10ratio between uracil and the abasic dodecamer oligonucleotide. Theuracil retention time was 2.43 minutes and the abasic oligonucleotidewas 21.68 minutes. The solution was lyophilized and stored in thefreezer until further use.

D. Preparation of Oligonucleotide Containing Multiple Uridine Sites

In the manner of Example 1A the following oligonucleotide was preparedGAC AGA GGU AGG AGA AGU GA (SEQ ID NO: 5) wherein U represents a2'-deoxyuridine nucleotide. The oligonucleotide is treated in accordancewith the procedure of Example 1C resulting in an oligonucleotide of thesequence GAC AGA GGN AGG AGA AGN GA (SEQ ID NO: 6) wherein N representsan abasic site within the oligonucleotide.

Example 2

Preparation of an Abasic Site Containing Oligonucleotide via a AbasicSugar Precursor

A. Preparation of5-O-4,4'-Dimethoxytrityl-1,2-Dideoxy-1-(o-nitrobenzoyl)-D-Ribofuranose-3-O-(2-Cyanoethyl-N,N'-Diisopropyl)Phosphoramidite.

5-O-4,4'-dimethoxytrityl-1,2-dideoxy-D-ribofuranose-3-O-(2-cyanoethyl-N,N'-diisopropyl)phosphoramidite is prepared in accordance with modification of theprocedures of Lyer, et al. Nucleic Acids Research 18: 2855 (1990) andDidier, et al., Tetrahedron Letters 32: 207 (1991) incorporated byreference herein in their entireties.

B. Preparation of Oligonucleotide Containing Abasic Site

Oligonucleotide having the sequence CGC AGN CAG CC wherein N representsan abasic site (SEQ ID NO:4) from Example 1C can be prepared inaccordance with modifications of the procedures of Lyer, et al. NucleicAcids Research 18: 2855 (1990) and Didier, et al., Tetrahedron Letters32: 207 (1991). In accordance with these procedures, an o-nitrobenzyldeoxyfuranose containing oligonucleotide is synthesized using theoligonucleotide synthetic methods of Lyer, et al. and Didier, et al.Photolysis utilizing a high intensity Hg lamp (300 nm) generates thecorresponding abasic site containing oligonucleotide. Such abasicoligonucleotides are also described in Horn, et al., Nucleosides andNucleotides 10:299 (1991).

Example 3

Preparation of Modified Abasic Sugar Precursors

A. Preparation of5-O-(4,4'-Dimethoxytrityl)-2-O-Methyl-1,2-Dideoxy-D-Ribofuranose-3-O-(2-Cyanoethyl-N,N'-Diisopropyl)Phosphoramidite.

1-O-methyl-D-ribofuranose is 3,5 protected with TIPS-Cl₂. It is then2-position methylated with either diazomethane or methyl iodide/silveroxide (CH₃ I/Ag₂ O). The composition is then treated with an aceticanhydride/acetic acid/sulfuric acid mixture to give a 1-O-acetyl,2-O-methyl 3,5 protected sugar. The 1-O-acetyl, 2-O-methyl 3,5 protectedsugar is deprotected with tetrabutyl ammonium fluoride, 5-positiondimethoxytritylated, and 3-position phosphitylated. Thereafter, thisphosphoramidite may be incorporated into an oligonucleotide by standardphosphoramidite procedures and ammonia deprotected to form a2'-O-methyl, 1' abasic site containing oligonucleotide.

B. Preparation of5-O-4,4'-Dimethoxytrityl-2-O-Methyl-1,2-Dideoxy-1-(o-nitrobenzoyl)-D-Ribofuranose-3-O-(2-Cyanoethyl-N,N'-Diisopropyl)Phosphoramidite.

1-O-acetyl 2,3,5-tri-O-benzoyl-D-ribofuranose is condensed witho-nitrobenzyl alcohol under Vorbruggen conditions. The resultant1-O-(ortho-nitrobenzyl)-2,3,5-tri-O-benzoyl (α,δ)-D-ribofuranose isdeprotected with ammonia and subsequently treated with TIPS-Cl₂. Theresultant 3,5-silyl protected 1-O-(ortho-nitro benzyl) D-ribofuranose isreacted with diazomethane or CH₃ I/Ag₂₀ to give the required 2-O-methylcompound. Subsequent3,5-deprotection, 5-dimethoxytritylation and3-phosphitylation gives the named phosphoramidite. The phosphoramiditecan be incorporated into an oligonucleotide via standard phosphoramiditeprocedures.

C. Preparation of5-O-(4,4'-Dimethoxytrityl)-2-Fluoro-1,2-Dideoxy-D-Ribofuranose-3-O-(2-Cyanoethyl-N,N'-Diisopropyl)Phosphoramidite.

1-O-(ortho-nitrobenzyl)-2,3,5-tri-O-benzoyl-D-ribofuranose isdeprotected at 2,3,5 positions using ammonia. Tritylation with excesstrityl chloride/pyridine/4-dimethylaminopyridine gives3-5-ditrityl-1-O-nitrobenzyl-D-ribo furanose. Oxidation at 2 positionwith CrO₃ followed by NaBH₄ reduction inverts the configuration at 2position yielding an arabino sugar. The arabino sugar is converted toits triflate at 2 position and the triflate is displaced with fluorideion to yield the 2-fluoride modified sugar which can be 5 positionprotected and phosphitylated to incorporate the sugar into anoligonucleotide via standard oligonucleotide synthesis.

Example 4

Oligonucleotides conjugated in the following example are set forth inTable 2.

                  TABLE II                                                        ______________________________________                                        OLIGOMER                                                                      (SEQ ID NO.)                                                                           TARGET   SEQUENCE     LINKER (L)                                     ______________________________________                                        A (SEQ ID                                                                              ICAM     TGG GAG CCA  3-carbon                                                                              P = S                                  NO: 7)            TAG CGA GGC-L                                                                              amino                                          B (SEQ ID                                                                              ICAM     TGG GAG CCA  3-carbon                                                                              P = O                                  NO: 7)            TAG CGA GGC-L                                                                              amino                                          C (SEQ ID                                                                              BPV      CTG TCT CCA* 2'amino-                                                                              P = O                                  NO: 8)            TCC TCT TCA CT                                                                             pentoxy                                        D (SEQ ID                                                                              BPV      CTG TCT CCA  3-carbon                                                                              P = O                                  NO: 9)            TCC TCT TCA  amino                                                            CT-L                                                        E (SEQ ID                                                                              BPV      CTG TCT CCA  6-carbon                                                                              P = O                                  NO: 9)            TCC TCT TCA  amino                                                            CT-L                                                        F (SEQ ID                                                                              CMV      GGC GUC UCC          2'-OMe                                 NO: 10)           AGG CGA UCU                                                                   GAC*                                                        G (SEQ ID                                                                              ICAM     TCT GAG TAG          2'-OMe                                 NO: 11)           CAG AGG AGC                                                                   TC*                                                         H (SEQ ID         GGA UGG CGU          2'-OMe                                 NO: 12)           CUC CAG GCG                                                                   AUC*                                                        I (SEQ ID         GGA UGG CGU  3-carbon                                                                              2'-OMe                                 NO: 13)           CUC CAG GCG  amino                                                            AUC-L                                                       J (SEQ ID         GGA UGG CGU  6-carbon                                                                              2'-OMe                                 NO: 13)           CUC CAG GCG  amino                                                            AUC-L                                                       K (SEQ ID         F-TGG GAG CCA                                                                              3-carbon                                                                              2'-OMe                                 NO: 7)            TAG CGA GGC-L                                                                              amino                                          ______________________________________                                         A* = 2O-aminopentoxy-2deoxyadenosine                                          C* = 2aminopropoxy cytosine                                                   F = Fluoroscein                                                          

A. 3' Terminus Polyamine End Labeled Oligonucleotide

1. 3'-Terminus Polyamine Oligonucleotide I

Polyamines were attached to the 3'-terminus end of a phosphodiesteroligonucleotide having the sequence D-polyamine (SEQ ID NO:9)-polyamine!, wherein the polyamine is one of the following:

                  TABLE III                                                       ______________________________________                                        1,6 Diaminohexane    Oligomer D (i)                                           Diethylenetriamine   Oligomer D (ii)                                          Triethylenetetramine Oligomer D (iii)                                         Spermine             Oligomer D (iv)                                          Pentaethylenehexamine                                                                              Oligomer D (v)                                           ______________________________________                                    

a. Preparation of the Intermediate Linker

The oligonucleotide sequence having a 3'-terminus amino group wassynthesized using 3'-amino modifier (with a three carbon linker)controlled pore glass (CPG) from Glen Research as the solid support. Thesynthesis was conducted with an Applied Biosystems 380B or 994 in the"Trityl-Off" mode. The resultant oligonucleotide was cleaved from thesolid support and deprotected with concentrated NH₄ OH for 16 hrs at 55°C. Purification on a Sephadex G-25 column yielded a 3'-amino modifiedoligonucleotide of the specified sequence.

b. Preparation of Polyamine Functionalized Oligonucleotide

The crude 3'-aminolinker-oligonucleotide (SEQ ID NO:9) (15 O.D. units,approximately 85 nmols) was dissolved in freshly prepared NaHCO₃ buffer(150 ul, 0.2M, pH 8.1) and treated with a solution of disuccinimidylsuberate (DSS) (approximately 5 mgs) dissolved in 150 ul of methylsulfoxide (DMSO). The reaction mixture was left to react for 20 minutesat room temperature. The mixture was then passed over a Sephadex G-25column (0.7×45 cm) to separate the activated oligonucleotide-DSS fromthe excess DSS. The oligonucleotide-DSS was then frozen immediately andlyophilized to dryness. A solution of polyamine in 0.33M NaOAc(approximately 6 mg polyamine in 300 ul 0.33M NaOAc, pH 5.2, finalsolution pH 6-8.0) was added to the dried oligonucleotide-DSS, and thismixture was allowed to react overnight at room temperature. Theresulting polyamine-oligonucleotide conjugate was characterized byreverse phase HPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA,solvent B was CH₃ CN. The HPLC gradient was from 0-10 mins, 95% solventA, 5% solvent B; linear increase to 40% solvent B in the next 50 minutesusing a Water's Delta-Pak C-18 reverse phase column. HPLC retentiontimes were as set forth in Table 4.

                  TABLE IV                                                        ______________________________________                                        Oligomer       Retention Time                                                 ______________________________________                                        unreacted D    26.44 mins                                                     Oligomer D (i) 27.48 mins                                                     Oligomer D (ii)                                                                              27.23 mins                                                     Oligomer D (iii)                                                                             27.27 mins                                                     Oligomer D (iv)                                                                              27.54 mins                                                     Oligomer D (v) 27.36 mins                                                     ______________________________________                                    

In a second test run under the same conditions the HPLC gradient was0-10 mins, 95% solvent A, 5% solvent B; linear increase to 15% solvent Bin 60 minutes. HPLC retention times were as set forth in Table 5.

                  TABLE V                                                         ______________________________________                                        Oligomer       Retention Time                                                 ______________________________________                                        unreated D     60.74 mins                                                     Oligomer D (ii)                                                                              62.37 mins                                                     Oligomer D (v) 65.24 mins                                                     ______________________________________                                    

Gel analysis showed progressively slower migration times for thepolyamine conjugates (the larger the polyamine, the slower themigration) versus the oligonucleotide alone. (Gel: 313-107)

c. Nuclease stability of 3' polyamine conjugates in Fetal Calf Serum

Polyamine conjugates of the invention are assessed for their resistanceto serum nucleases by incubation of the oligonucleotides in mediacontaining various concentrations of fetal calf serum. Labeledoligonucleotides are incubated for various times, treated with proteaseK and then analyzed by gel electrophoresis on 20% polyacrylamide-ureadenaturing gels and subsequent autoradiography or phosphor-imaging.Autoradiograms are quantitated by laser densitometry. Based upon thelocation of the modifications and the known length of theoligonucleotide it is possible to determine the effect of the particularmodification on nuclease degradation. For the cytoplasmic nucleases, aHL60 cell line is used. A post-mitochondrial supernatant is prepared bydifferential centrifugation and the labeled oligonucleotides areincubated in this supernatant for various times. Following theincubation, oligonucleotides are assessed for degradation as outlinedabove for serum nucleolytic degradation. Autoradiography results arequantitated for comparison of the unmodified and the modifiedoligonucleotides. The t_(1/2) are set forth below.

                  TABLE VI                                                        ______________________________________                                        Oligonucleotide    t.sub.1/2 (hours)                                          ______________________________________                                        wild type oligomer D                                                                             0.5 (no aminolinker)                                       unreacted oligomer D                                                                             22 (with aminolinker)                                      oligomer D (ii)    48                                                         oligomer D (v)     >50                                                        ______________________________________                                    

2. 3'-Terminus Polyamine Conjugate II

Polyamines were attached to the 3'-terminus end of a phosphodiesteroligonucleotide having the sequence E-polyamine (SEQ ID NO:9)-polyamine! wherein the polyamine is one of the following:

                  TABLE VII                                                       ______________________________________                                        Diethylenetriamine    Oligomer E (i)                                          Pentaethylenehexamine Oligomer E (ii)                                         ______________________________________                                    

a. Preparation of the Intermediate Linker

The intermediate linker was prepared as described in Example 4-A-1-asubstituting a 3' amino modifier with a six carbon linker (Clonetech,Palo Alto, Calif.) for the 3'-amino modifier (with a three carbonlinker.

3. Preparation of Polyamine Functionalized Oligonucleotide

The polyamine functionalized oligonucleotide was prepared in accordancewith Example 4-A-1-b. The resulting polyamine-oligonucleotide conjugatewas characterized by reverse phase HPLC and a 20% denaturing gel.Solvent A was 50 mM TEAA, solvent B was CH₃ CN. The HPLC gradient wasfrom 0-10 mins, 95% solvent A, 5% solvent B; linear increase to 25%solvent B in the next 50 minutes using a Water's Delta-Pak C-18 reversephase column. HPLC retention times were as set forth in Table 8.

                  TABLE VIII                                                      ______________________________________                                        Oligomer       Retention Time                                                 ______________________________________                                        unreated E     41.38 mins                                                     Oligomer E (i) 43.29 mins                                                     Oligomer E (ii)                                                                              43.43 mins                                                     ______________________________________                                    

Gel analysis showed progressively slower migration times for thepolyamine conjugates (the larger the polyamine, the slower themigration) versus the oligonucleotide alone. (Gel: 353-35).

4. 3' -Terminus Polyamine Conjugate III

Polyamines were attached to the 3'-terminus end of a phosphorothioateoligonucleotide having the sequence A-polyamine (SEQ ID NO:7)-polyamine!where the polyamine is one of the following:

                  TABLE IX                                                        ______________________________________                                        1,6 Diaminohexane    Oligomer A (i)                                           Diethylenetriamine   Oligomer A (ii)                                          Triethylenetetramine Oligomer A (iii)                                         Spermine             Oligomer A (iv)                                          Pentaethylenehexamine                                                                              Oligomer A (v)                                           ______________________________________                                    

a. Preparation of the Intermediate Linker

The intermediate linker was prepared as described in Example 4-A-1-autilizing the Beaucage reagent (3H-1,2-benzodithioate-3-one 1,1-dioxide,see Radhakrishnan, P. I., Egan, W., Regan, J. B. and Beaucage, S. L.,(1990) J. Am. Chem. Soc., 112:1253) to form the phosphothioateinternucleotide backbone. The 3'-aminolinker was introduced as describedin example 4-A-1-a.

b. Preparation of Polyamine Functionalized Oligonucleotide

Oligonucleotides were functionalized as described in Example 4-A-1-b.The resulting polyamine-oligonucleotide conjugate was characterized byreverse phase HPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA,solvent B was CH₃ CN. The HPLC gradient was from 0-10 mins, 95% solventA, 5% solvent B; linear increase to 40% solvent B in the next 50 minutesusing a Water's Delta-Pak C-18 reverse phase column. HPLC retentiontimes were as set forth in Table 10.

                  TABLE X                                                         ______________________________________                                        Oligomer       Retention Time                                                 ______________________________________                                        unreacted A    30.77 mins                                                     Oligomer A (iii)                                                                             31.31 mins                                                     Oligomer A (v) 31.02 mins                                                     ______________________________________                                    

In a second test run under the same conditions, the HPLC gradient was0-10 mins, 95% solvent A, 5% solvent B; linear increase to 15% solvent Bin 60 minutes. Retention times were as set forth in Table 11.

                  TABLE XI                                                        ______________________________________                                        Oligomer       Retention Time                                                 ______________________________________                                        unreated A     68.62 mins                                                     Oligomer A (i) 68.70 mins                                                     Oligomer A (ii)                                                                              68.69 mins                                                     ______________________________________                                    

In a second test run under the same conditions, HPLC retention timeswere as set forth in Table 12.

                  TABLE XII                                                       ______________________________________                                        Oligomer       Retention Time                                                 ______________________________________                                        unreated A     30.34 mins                                                     Oligomer A (iv)                                                                              30.57 mins                                                     Oligomer A (v) 30.72 mins                                                     ______________________________________                                    

Gel analysis showed progressively slower migration times for thepolyamine conjugates (the larger the polyamine, the slower themigration) versus the oligonucleotide alone. (Test run 1 Gel, 313-82;Test run 2 Gel, 285-138; Test run 3 Gel, 353-57)

C. Preparation of Biotin Functionalized Oligonucleotide PolyamineConjugate

To further characterize the oligonucleotide polyamine conjugate, biotinwas attached to the free amines made available by the polyaminesattached in Example 4-A-4-b. About 10 O.D. units (A₂₆₀) of OligomersA(i) and A(ii) (approximately 58 nmoles) were dried in a microfuge tube.The oligonucleotide polyamine conjugate was rehydrated in 400 ul of 0.2MNaHCO₃ (pH 8.1) buffer and D-biotin-N-hydroxysuccinimide ester(approximately 5.0 mgs biotin for the 1,6 Diaminohexane conjugate, 8.0mgs for the Diethylenetriamine) (Sigma) was added followed by 200 ul ofDMF. The solution was left to react overnight at room temperature. Thesolution was then passed over a NAP-25 column and analyzed by reversephase HPLC. Solvent A was 50 mM TEAA and solvent B was CH₃ CN. The HPLCgradient was 0-10 mins, 95% A, 5% B; linear increase to 40% B in thenext 50 minutes using a Water's Delta-Pak C-18, reverse phase column.The HPLC retention times were as set forth in Table 13.

                  TABLE XIII                                                      ______________________________________                                        Oligomer         Retention Time                                               ______________________________________                                        unreated A       30.77 mins                                                   Oligomer A (i)   31.31 mins                                                   Oligomer A (i)-Biotin                                                                          35.56 mins                                                   Oligomer A (ii)  31.02 mins                                                   Oligomer A (ii)-Biotin                                                                         36.23 mins                                                   ______________________________________                                    

5. 3' -Terminus Polyamine Conjugate IV

Polyamines were attached to the 3'-terminus end of the phosphodiesteroligonucleotide having the sequence B-polyamine (SEQ ID NO:7)-polyamine! wherein the polyamine is one of the following:

                  TABLE XIV                                                       ______________________________________                                        Diethylenetriamine   Oligomer B (i)                                           Triethylenetetramine Oligomer B (ii)                                          Spermine             Oligomer B (iii)                                         Pentaethylenehexamine                                                                              Oligomer B (iv)                                          ______________________________________                                    

a. Preparation of the Intermediate Linker

The intermediate linker was prepared as described in Example 4-A-1-a.

b. Preparation of Polyamine Functionalized Oligonucleotide

The oligonucleotide was functionalized with polyamines as described inExample 4-A-1-b. The resulting polyamine-oligonucleotide conjugate wascharacterized by reverse phase HPLC and a 20% denaturing gel. Solvent Awas 50 mM TEAA, solvent B was CH₃ CN. The HPLC gradient was from 0-10mins, 95% solvent A, 5% solvent B; linear increase to 40% solvent B inthe next 50 minutes using a Water's Delta-Pak C-18 reverse phase column.HPLC retention times were as set forth in Table 15.

                  TABLE XV                                                        ______________________________________                                        Oligomer       Retention Time                                                 ______________________________________                                        untreated B    25.71 mins                                                     Oligomer B (i) 26.11 mins                                                     Oligomer B (ii)                                                                              25.26 mins                                                     Oligomer B (iii)                                                                             25.10 mins                                                     Oligomer B (iv)                                                                              25.12 mins                                                     ______________________________________                                    

Gel analysis showed progressively slower migration times for thepolyamine conjugates (the larger the polyamine, the slower themigration) versus the oligonucleotide alone. (Gel: 313-112)

B. 2' Internal Polyamine Labeled Oligonucleotide

1. 2'-Internal Polyamine oligonucleotide I

Polyamines were attached to the 2'-internal linker site of aphosphodiester oligonucleotide having the sequence C-polyamine (SEQ IDNO: 8)-polyamine! wherein the polyamine is one of the following:

                  TABLE XVI                                                       ______________________________________                                        Diethylenetriamine   Oligomer C (i)                                           Triethylenetetramine Oligomer C (ii)                                          Pentaethylenehexamine                                                                              Oligomer C (iii)                                         ______________________________________                                    

a. Preparation of the Intermediate Linker

The intermediate linker was prepared as described in Example 4-A-1-aincorporating a modified adenosine phosphoramidite (with a2'-aminolinker) at position #9. This oligonucleotide and the 2'-aminolinker have been described in Manoharan, M., Guinosso, C. J., Cook, P.D., Tetrahedron Letters, 32, (pgs. 7171-7174), 1991.

b. Preparation of Polyamine Functionalized Oligonucleotide

The oligonucleotide was functionalized as described in Example 4-A-1-b.The resulting polyamine-oligonucleotide conjugate was characterized byreverse phase HPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA,solvent B was CH₃ CN. The HPLC gradient was from 0-10 mins, 95% solventA, 5% solvent B; linear increase to 40% solvent B in the next 50 minutesusing a Water's Delta-Pak C-18 reverse phase column. HPLC retentiontimes were as set forth in Table 17.

                  TABLE XVII                                                      ______________________________________                                        Oligomer       Retention Time                                                 ______________________________________                                        untreated C    26.20 mins                                                     Oligomer C (i) 27.52 mins                                                     Oligomer C (ii)                                                                              27.50 mins                                                     Oligomer C (iii)                                                                             27.59 mins                                                     ______________________________________                                    

Gel analysis showed progressively slower migration times for thepolyamine conjugates (the larger the polyamine, the slower themigration) versus the oligonucleotide alone. (Gel: 313-97)

C. 3' Terminus Polyamine End Labeled Oligonucleotide, Using a2'-aminolinker

1. 3' Terminus Polyamine Labeled Oligonucleotide I, Using a2'-aminolinker

Polyamines were attached to the 3'-terminus end of a phosphodiester(2'-OMe) oligonucleotide via a 2'-aminolinker having the sequenceF-polyamine (SEQ ID NO:10)-polyamine! wherein the polyamine ispentaethylenehexamine (oligomer F(i)).

a. Preparation of the Intermediate Linker

The intermediate linker was prepared as described in Example 4-A-1-a,except that a modified cytosine CPG (with a 2'-propylaminolinker) wasintroduced at the 3' end. The 2'-modification can be prepared bymodification of the procedure previously described in application Ser.No. 918,362 filed Jul. 23, 1992. The CPG containing2'-w-phthalimido-propoxy-cytidine was synthesized according to thestandard protocols reported in the literature. See, for example, B. S.Sproat and A. I. Lamond, in "Oligonucleotides and Analogues" edited byF. Eckstein , IRL Press at Oxford University Press (1991) p71-72.

b. Preparation of Polyamine Functionalized Oligonucleotide

The polyamine functionalized oligonucleotide was prepared in accordancewith Example 4-A-1-b. The resulting polyamine-oligonucleotide conjugatewas characterized by reverse phase HPLC and a 20% denaturing gel.Solvent A was 50 mM TEAA, solvent B was CH₃ CN. The HPLC gradient wasfrom 0-10 mins, 95% solvent A, 5% solvent B; linear increase to 40%solvent B in the next 50 minutes using Water's Delta-Pak C-18 reversephase column. HPLC retention times were as set forth in Table 18.

                  TABLE XVIII                                                     ______________________________________                                        Oligomer       Retention Time                                                 ______________________________________                                        unreacted F    28.53 mins                                                     oligomer F (i) 29.47 mins                                                     ______________________________________                                    

Gel analysis showed progressively slower migration times for thepolyamine conjugate versus the oligonucleotide alone. (Gel: 397-85)

2. 3' Terminus Polyamine Labeled Oligonucleotide II, Using a2'-aminolinker

Polyamines were attached to the 3'-terminus end of a phosphodiester(2'-OMe) oligonucleotide via a 2'-aminolinker having the sequenceG-polyamine (SEQ ID NO:11)-polyamine! wherein the polyamine ispentaethylenehexamine (oligomer G(i)).

a. Preparation of the Intermediate Linker

The intermediate linker was prepared in accordance with the methoddescribed in Example 4-A-1-a.

b. Preparation of Polyamine Functionalized Oligonucleotide

The polyamine functionalized oligonucleotide was prepared in accordancewith the procedures described in Example 4-A-1-b. The resultingpolyamine-oligonucleotide conjugate was characterized by reverse phaseHPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B wasCH₃ CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solventB; linear increase to 40% solvent B in the next 50 minutes using Water'sDelta-Pak C-18 reverse phase column. HPLC retention times were as setforth in Table 19.

                  TABLE XIX                                                       ______________________________________                                        Oligomer       Retention Time                                                 ______________________________________                                        unreacted G    28.43 mins                                                     oligomer G (i) 29.06 mins                                                     ______________________________________                                    

Gel analysis showed progressively slower migration times for thepolyamine conjugate versus the oligonucleotide alone. (Gel: 397-85)

3. 3' Terminus Polyamine Labeled Oligonucleotide III Using a2'-aminolinker

Polyamines were attached to the 3'-terminus end of a phosphodiester(2'-OMe) oligonucleotide via a 2'-aminolinker having the sequenceH-polyamine (SEQ ID NO:12)-polyamine! wherein the polyamine ispentaethylenehexamine.

a. Preparation of the Intermediate Linker

The intermediate linker is prepared in accordance with methods describedin Example 4-A-1-a.

b. Preparation of Polyamine Functionalized Oligonucleotide

The polyamine functionalized oligonucleotide is prepared in accordancewith methods described in Example 4-A-1-b. The resultingpolyamine-oligonucleotide conjugate was characterized by reverse phaseHPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B wasCH₃ CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solventB; linear increase to 40% solvent B in the next 50 minutes using Water'sDelta-Pak C-18 reverse phase column. HPLC retention times were as setforth in Table 20.

                  TABLE XX                                                        ______________________________________                                        Oligomer       Retention Time                                                 ______________________________________                                        unreacted H    28.49 mins                                                     oligomer H (i) 30.36 mins                                                     ______________________________________                                    

Gel analysis showed progressively slower migration times for thepolyamine conjugate versus the oligonucleotide alone. (Gel: 397-85)

Example 5

Polyamine Labeled 2'-OMe Oligonucleotides and Other RNA Mimics

1. Polyamine Labeled 2'-OMe Oligonucleotide I

Polyamines were attached to the 3'-terminus end of a phosphodiester(2'-OMe) oligonucleotide (via a 3 carbon linker) having the sequenceI-polyamine (SEQ ID NO:13)-polyamine! wherein the polyamine ispentaethylenehexamine (oligomer I(i)).

a. Preparation of the Intermediate Linker

The intermediate linker is prepared in accordance with methods describedin Example 4-A-1-a.

b. Preparation of Polyamine Functionalized Oligonucleotide

The polyamine functionalized oligonucleotide is prepared in accordancewith methods described in Example 4-A-1-b. The resultingpolyamine-oligonucleotide conjugate was characterized by reverse phaseHPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B wasCH₃ CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solventB; linear increase to 40% solvent B in the next 50 minutes using Water'sDelta-Pak C-18 reverse phase column. HPLC retention times were as setforth in Table 21.

                  TABLE XXI                                                       ______________________________________                                        Oligomer       Retention Time                                                 ______________________________________                                        unreacted I    28.93 mins                                                     oligomer I (i) 29.59 mins                                                     ______________________________________                                    

Gel analysis showed progressively slower migration times for thepolyamine conjugate versus the oligonucleotide alone. (Gel: 353-156)

2. Polyamine Labeled 2'-OMe Oligonucleotide II

Polyamines were attached to the 3'-terminus end of a phosphodiester(2'-OMe) oligonucleotide (via a 6 carbon linker) having the sequenceJ-polyamine (SEQ ID NO:13)-polyamine! wherein the polyamine ispentaethylenehexamine (oligomer J(i)).

a. Preparation of the Intermediate Linker

The intermediate linker is prepared in accordance with methods describedin Example 4-A-1-a.

b. Preparation of Polyamine Functionalized Oligonucleotide

The polyamine functionalized oligonucleotide is prepared in accordancewith methods described in Example 4-A-1-b. The resultingpolyamine-oligonucleotide conjugate was characterized by reverse phaseHPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B wasCH₃ CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solventB; linear increase to 40% solvent B in the next 50 minutes using Water'sDelta-Pak C-18 reverse phase column. HPLC retention times were as setforth in Table 22.

                  TABLE XXII                                                      ______________________________________                                        Oligomer       Retention Time                                                 ______________________________________                                        unreacted J    28.76 mins                                                     oligomer J (i) 29.39 mins                                                     ______________________________________                                    

Gel analysis showed progressively slower migration times for thepolyamine conjugate versus the oligonucleotide alone. (Gel: 397-85)

3. Polyamine Labeled 2'-OMe oligonucleotide III

Polyamines were attached to the 3'-terminus end of a phosphodiester(2'-OMe) oligonucleotide (via a 3 carbon linker) having another reportergroup (such as biotin, fluorescein) at the other end in the sequenceK-polyamine (SEQ ID NO:7)-polyamine!. Fluoroscein at 5' end was addedusing the required amidite commercially available from Clontech. Thepolyamine is one of the following

pentaethylenehexamine oligomer K(i)

spermine oligomer K(ii)

a. Preparation of the Intermediate Linker

The intermediate linker is prepared in accordance with methods describedin Example 4-A-1-a.

b. Preparation of Polyamine Functionalized Oligonucleotide

The polyamine functionalized oligonucleotide is prepared in accordancewith methods described in Example 4-A-1-b. The resultingpolyamine-oligonucleotide conjugate was characterized by reverse phaseHPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B wasCH₃ CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solventB; linear increase to 40% solvent B in the next 50 minutes using Water'sDelta-Pak C-18 reverse phase column. HPLC retention times were as setforth in Table 23.

                  TABLE XXIII                                                     ______________________________________                                        Oligomer       Retention Time                                                 ______________________________________                                        unreacted K    31.35 mins                                                     oligomer K (i) 31.96 mins                                                     oligomer K (ii)                                                                              32.15 mins                                                     ______________________________________                                    

Gel analysis showed progressively slower migration times for thepolyamine conjugate versus the oligonucleotide alone. (Gel: 353-149)

Example 6

5' Terminus Polyamine End Labeled Oligonucleotide

1. 5'-Terminus Polyamine Oligonucleotide I

Polyamines were attached to the 5'-terminus end of a phosphodiesteroligonucleotide having the following sequences:

5'-aminolinker-TCAG (oligomer L)

5'-aminolinker-CGCACGC (oligomer M) to provide the polyamineoligonucleotides:

5'-polyamine-TCAG (oligomer L(i))

5'-polyamine-CGCACGC (oligomer M(i)) wherein the polyamine ispentaethylenehexamine.

a. Preparation of the Intermediate Linker

The oligonucleotide sequence having a 5'-terminus amino group wassynthesized using Aminolink-II(with a six carbon linker) phosphoramiditefrom Applied Biosystems in the last round of synthesis. The synthesiswas conducted with an Applied Biosystems 380B or 994 in the "Trityl-On"mode. The resultant oligonucleotide was cleaved from the solid supportand deprotected with concentrated NH₄ OH for 16 hrs at 55° C.Purification on a Sephadex G-25 column yielded a 5'-amino modifiedoligonucleotide of the specified sequence.

b. Preparation of Polyamine Functionalized Oligonucleotide L(i)

The crude 5'-aminolinker-oligonucleotide (150 O.D. units, approximately3.75 mmols) was dissolved in freshly prepared NaHCO₃ buffer (900 ul,0.2M, pH 8.1) and treated with a solution of disuccinimidyl suberate(DSS) (approximately 30 mgs) dissolved in 750 ul of methyl sulfoxide(DMSO). The reaction mixture was left to react for 20 minutes at roomtemperature. The mixture was divided into three portions and then passedover a Sephadex G-25 column (0.7×45 cmx3columns) to separate theactivated oligonucleotide-DSS from the excess DSS. Theoligonucleotide-DSS was then frozen immediately and lyophilized todryness. A solution of polyamine in 0.33M NaOAc (approximately 60 mLpolyamine in 1950 ul 0.33M NaOAc, pH 5.2, final solution pH 6-8.0) wasadded to the dried oligonucleotide-DSS, and this mixture was allowed toreact overnight at room temperature.

c. Preparation of Polyamine Functionalized Oligonucleotide M(i)

The crude 5'-aminolinker-oligonucleotide (oligomer M) (150 O.D. units,approximately 2.50) was reacted as described in Example 6(b).

d. Characterization of 5' Polyamine Functionalized Oligonucleotides

The resulting polyamine-oligonucleotide conjugates were characterized byreverse phase HPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA,solvent B was CH₃ CN. The HPLC gradient was from 0-10 mins, 95% solventA, 5% solvent B; linear increase to 40% solvent B in the next 50 minutesusing Water's Delta-Pak C-18 reverse phase column. HPLC retention timeswere as set forth in Table 24.

                  TABLE XXIV                                                      ______________________________________                                                DNA TARGET   RNA TARGET                                                         T.sub.m                                                                              ΔT.sub.m                                                                       ΔΔG.sup.o.sub.37°C.                                               (°C.)                                                                       (°C.)                                                                       ΔΔG.sup.o.sub.37.degr                                             ee.C.                                ______________________________________                                        Oligomer  (°C.)                                                                         (°C.)                                                                         ΔΔG.sup.o.sub.37°C.                                               (°C.)                                                                       (°C.)                                                                       ΔΔG.sup.o.sub.37.degr                                             ee.C.                                ______________________________________                                        wild type 60.6   --     --     64.9 --   --                                   oligomer D                                                                    oligomer D                                                                              60.3   -0.3   +0.3   64.6 -0.3 0.0                                  oligomer D +                                                                            60.8   +0.2   0.0    65.1 +0.2 0.0                                  5'-6-carbon                                                                   amino linker)                                                                 oligomer E                                                                              60.8   +0.2   -0.8   65.8 +0.9 -1.0                                 oligomer E(i)                                                                           61.2   +0.6   -1.4   66.3 +1.4 -1.9                                 oligomer E +                                                                            61.5   +0.9   -1.7   67.1 +2.2 -2.1                                 spermine                                                                      oligomer E(ii)                                                                          61.2   +0.6   -1.3   67.5 +2.6 -2.6                                 ______________________________________                                    

Example 7

Preparation of a Reactive Site containing Oligonucleotide

An oligonucleotide having the sequence TGGGAGCCATAGCGAGGUCT (SEQ ID NO:14) is treated with uracil DNA glycosylase followed by T4 endonuclease.The product is then treated with 1-phthalimidobutyl-4-thiol.Nucleophilic attack by the thiol adds to the protected aminobutyl moietyto what was the 3' position of the opened nucleotide. Treatment of thiscomposition with hydrazine will deblock the phthalimide yielding anamino species which is then treated with bifunctional linker followed bytreatment with an appropriate polyamine species as per Example 4-A-1-b.

Example 8

Preparation of Polyamine Conjugated Oligonucleotide

An oligonucleotide is prepared as described in Example 7 treating theproduct with NH₂ --CH₂ --CH₂ --SH. The thiol group will attack thedouble bond of the opened nucleotide. This species may then be furtherderivatized with a reactive group.

Example 9

Thermodynamic Parameters of Oligoamine-Oligonucleotide Conjugates withDNA and RNA Targets

The ability of the functionalized oligonucleotides of the invention tohybridize to their complementary RNA or DNA sequences is determined bythermal melting analysis. The RNA complement is synthesized from T7 RNApolymerase and a template-promoter of DNA synthesized with an AppliedBiosystems, Inc. 380B nucleic acid synthesizer. The RNA species ispurified by ion exchange using FPLC (LKB Pharmacia, Inc.) or bydenaturing urea-PAGE. Natural antisense oligonucleotides or thosecontaining functionalization at specific locations are added to eitherthe RNA or DNA complement at stoichiometric concentrations to formhybrid duplexes. The absorbance (260 nm) hyperchromicity dependence ontemperature upon duplex to random coil transition is monitored using aGilford Response II spectrophotometer. These measurements are performedin a buffer of 10 mM Na-phosphate, pH 7.4, 0.1 mM EDTA, and NaCl toyield an ionic strength of either 0.1M or 1.0M. Data are analyzed by agraphic representation of 1/T_(m) vs ln Ct!, where Ct! is the totaloligonucleotide concentration. From this analysis the thermodynamicparameters are determined. Based upon the information gained concerningthe stability of the duplex or hetero-duplex formed, the placement ofthe polyamines into oligonucleotides is assessed for its effects onhelix stability. Modifications that drastically alter the stability ofthe hybrid exhibit reductions or enhancements in the free energy (deltaG) and decisions concerning their usefulness in antisenseoligonucleotides are made.

Example 10

Conjugation of Polyamines to Abasics Sites Containing Oligonucleotides

To 15.2 ODS of an abasic oligonucleotide (SEQ ID NO: 4) in 100 μl waterwas added 25 μl 1M NaOAc (pH 5.0) solution. The final concentration ofthe acetate buffer was 0.2M. 5.3 mg of triethylenetetramine wasdissolved in 500 μl of 1M NaOAc (pH 5.0) solution. 50 μl of theresulting solution was added to the oligonucleotide solution followed by50 μl of NaCNBH₃ (57 MM solution). The pH of the resulting solution wasbelow 8.0. The solution was vortexed and left to stand overnight. HPLCand Gel analysis indicated conjugation of the triethylenetetramine tothe oligonucleotide. The conjugated oligonucleotide was purified by G-25and HPLC. HPLC retention times are set forth in Table 25.

                  TABLE XXV                                                       ______________________________________                                        Oligomer              Retention time (mins)                                   ______________________________________                                        parent oligonucleotide (SEQ ID NO: 3)                                                               26.66                                                   abasic oligonucleotide (SEQ ID NO: 4)                                                               26.16                                                   (SEQ ID NO: 4)-triethylenetetramine conjugate                                                       26.04                                                   ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 14                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TGGGAGCCATAGCGAGGC18                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 19                                                              (D) OTHER INFORMATION: /note= "abasic, aldehydic                              species"                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TGGGAGCCATAGCGAGGCN19                                                         (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "2'deoxyuridine                                 residue"                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CGCAGUCAGCC11                                                                 (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /note= "abasic residue"                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CGCAGNCAGCC11                                                                 (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "2'deoxyuridine                                 residue"                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 18                                                              (D) OTHER INFORMATION: /note= "2'deoxyuridine                                 residue"                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GACAGAGGUAGGAGAAGUGA20                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "abasic residue"                                (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 18                                                              (D) OTHER INFORMATION: /note= "abasic residue"                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GACAGAGGNAGGAGAAGNGA20                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TGGGAGCCATAGCGAGGC18                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note=                                                 "2'-O-aminopentoxy-2'-deoxyadenosine"                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CTGTCTCCATCCTCTTCACT20                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CTGTCTCCATCCTCTTCACT20                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 21                                                              (D) OTHER INFORMATION: /note= "2'-aminopropoxy                                cytosine"                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GGCGUCUCCAGGCGAUCUGAC21                                                       (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 20                                                              (D) OTHER INFORMATION: /note= "2'-aminopropoxy                                cytosine"                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TCTGAGTAGCAGAGGAGCTC20                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 21                                                              (D) OTHER INFORMATION: /note= "2'-aminopropoxy                                cytosine"                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GGAUGGCGUCUCCAGGCGAUC21                                                       (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GGAUGGCGUCUCCAGGCGAUC21                                                       (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      TGGGAGCCATAGCGAGGUCT20                                                        __________________________________________________________________________

What is claimed is:
 1. A compound having the structure: ##STR14##wherein R₄ is an oligonucleotide and M is a pendent group having apolyamine species attached thereto.
 2. The compound of claim 1 wherein Mis R₇ S or R₇ NH wherein R₇ is a polyamine species.
 3. The compound ofclaim 2 wherein the polyamine species comprises at least one nitrogenatom having a free electron pair.
 4. The compound of claim 1 furtherincluding one or more of reporter groups, alkylating agents,intercalating agents, cell receptor binding molecules, steroids,peptides, crown amines, porphyrins, or cross-linking agents attached toat least one of the nitrogen atoms of said polyamine.
 5. The compound ofclaim 1 in a pharmaceutically acceptable carrier.